ALIX_Bro1-V binding to different p6^Gag proteins. (A) Representative biosensor sensograms for ALIX_Bro1-V binding to immobilized wild-type (main graph) or Y45A mutant (inset) SIV_mac239 glutathione S-transferase (GST)-p6^Gag proteins. Samples were analyzed in triplicate. p6^Gag proteins, minimal binding peptides (blocked N and C termini), were created, and the binding studies were performed and analyzed as described previously (37). RU, response units. (B) Representative biosensor binding isotherms for ALIX_Bro1-V binding to SIV_mac239 GST-p6^Gag or SIV_agmTan-1 p6^Gag proteins. (C) Summary of ALIX_Bro1-V dissociation constants for different retroviral p6/p9 proteins/peptides. Values for SIV peptides are the means from two independent experiments (each measured in triplicate) ± ranges. Dissociation constants for the HIV-1 and EIAV peptides were reported previously (37) but are reprinted here for reference.

Primary sequences and functional analyses of the late domains within primate lentiviral p6^Gag proteins. (A) Maximum likelihood phylogenetic tree showing the different primate lentiviral lineages and their p6^Gag proteins (drawn to scale as white boxes). TSG101-binding P(S/T)AP late domains are shown in blue, and putative ALIX-binding late domains are shown in red (predicted ALIX contact residues) or black (solvent exposed residues). ALIX-binding-site types are designated at right (see text for explanation). Sequences that most closely match the crystallographically characterized ALIX complexes are denoted with stars. Consensus sequences for the designated lineage(s) were derived from reference 19, and residues conserved at >85% identity are shown in capital letters. p6^Gag proteins from the HIV-1/SIV_cpz lineage fall into three different classes that either have type 1 or 2 ALIX-binding sites or lack apparent ALIX-binding sites (and also lack tyrosines). Putative ALIX-binding sites within SIV_wrc proteins can have either a Trp or Tyr residue in the second position, and the effect of the Trp substitution on ALIX binding has not been tested. The scale bar represents 0.1 substitution per site, and the tree was adapted from reference 1a. (B) Mutations in the SIV_mac251 p6^Gag ₁₁PTAP₁₄ (ΔPTAP) and ₃₈EKPYKEVTEDLLHL₅₁ (ΔYL) sequences inhibited virion release (Western blot, panel 1) and reduced viral titers (bottom graph, single-cycle infectivity assays), and ALIX overexpression stimulated release of the SIV_mac251 ΔPTAP construct. Cells were transfected with the designated SIV_mac251 vectors (WISP10-480-484), and cotransfected with either an empty pCI-neo vector control (lanes 1 to 4) or vectors expressing either wild-type (WT) FLAG-ALIX (lanes 5 to 8) or the designated mutant (lanes 9 and 10) FLAG-ALIX proteins. Vector transduction titers shown in the graph were measured in single-cycle infectivity assays (n = 5 assays; values are shown plus standard deviations). Western blots showing the levels of cell-associated CA and ALIX are also shown (CELL), with endogenous ALIX expression levels (lanes 1 to 4) enhanced 20-fold relative to exogenous ALIX expression levels (lanes 5 to 10) for ease of visualization.

Structures of SIV late domains bound to the second V-domain arm of human ALIX_Bro1-V. (A) SIV_mac239 p6^Gag late-domain peptide (magenta sticks) and human ALIX_Bro1-V protein, represented as a blue ribbon and surface, with the side chains of binding-site residues shown explicitly and labeled. The p6 tyrosine is indicated with a Y. The p6 peptide is oriented with the N terminus at the bottom of the figure. (B) SIV_agmTam-1 p6^Gag late-domain peptide and ALIX_Bro1-V complex. (C) Overlay of the ALIX in complex with late-domain peptides of HIV-1 p6^Gag (Protein Data Bank code 2R02; green), EIAV p9^Gag (Protein Data Bank code 2R03; turquoise), SIV_mac239 p6^Gag (magenta), and SIV_agmTan-1 p6^Gag (salmon). Panels A to C were generated using PyMOL (6). (D) Late-domain peptide sequences used for crystallography and binding studies, aligned on the basis of the crystal structures. Residues modeled in the crystal structures are in boldface, and those lacking electron density are in italics. Residues that are structurally equivalent in all four complexes are highlighted by a gray background. Residues that bury more than 50% of their solvent-accessible surface at the protein interface are underlined. Note that ALIX binds three different types of viral sequence motifs, which we have designated types 1 to 3.